For cell cycle transition from G/G to S phase, at least three types of molecular systems are involved in a concerted manner. These include: 1) the signal transduction pathway which receives extracellular signals and transmits these into the cell, 2) cyclins with associated kinases and modulators which may regulate passage through the G restriction point and other cell cycle check points, and 3) the metabolic processes required for doubling the essential cellular components including DNA.

While a variety of signaling systems work together to either induce or block proliferation, one of the best characterized, and perhaps the most universally required signaling systems for proliferation, involves proto-oncogenes including cellular Ras proteins. When growth factors bind to their tyrosine kinase receptors, a series of phosphorylations and resulting intermolecular interactions induce the activation of cellular Ras proteins(1) . Active Ras in turn binds to cellular Raf kinases resulting in their activation and ultimately the activation of mitogen-activated protein kinases (MAP ()kinases)(2) . The activated MAP kinases enter the nucleus and presumably stimulate the activity of genes required for proliferation (3, 4) .

Even though cellular proliferation in most cell types requires the activity of the above signal transduction system, the orderly transit of the growth factor-stimulated cell through the cell cycle depends upon the action of the second class of nuclear proteins, including cyclins, CDKs, and proteins which modulate the activity of the complexes they form. It is believed that cyclins and associated CDKs control progress through cell cycle phases. In so doing they would regulate the activity of the third group of molecules required for cell proliferation, those which catalyze the metabolism required to duplicate DNA and other critical cellular components necessary for cell division. For example, active cyclin D/CDK4 is known to phosphorylate the pRb(5, 6) . Hyperphosphorylation of the pRb results in the release of the transcription factor complex E2F/DP which is bound to and inactivated by hypophosphorylated pRb(7) . Active E2F/DP is known to induce the transcription of molecules required for DNA synthesis, such as dihydrofolate reductase(8) .

A full understanding of the control of cell cycle progression from G to S phase will require not only an understanding of these three separate processes essential for cell cycle progression (signaling molecules, cyclins and associated proteins, and enzymes required for DNA synthesis), but an understanding of how these classes of proteins interact with each other. As described above and elsewhere (9) , the molecular mechanism connecting the activity of cyclins and their associated proteins to DNA synthesis is well characterized. Of particular interest in this study is the interaction between proto-oncogene signaling molecules and cyclin-associated proteins. Direct evidence for such an interaction was obtained in a recent microinjection study. The proliferation of most normal cells is blocked following the microinjection of a neutralizing anti-Ras antibody(10) . When cells which have received such injections receive a subsequent injection of purified adenoviral E1A protein which is able to release E2F/DP from pRb(7, 11) , the cells are able to rapidly enter S phase with high efficiency(12) . Thus, the activity of E2F/DP, a normal consequence of cyclin-associated protein action, is able to compensate for blockage of the action of proto-oncogene signaling molecules. It therefore appears that Ras activity in the cell is required for entry into S phase to a certain extent based upon its ability to ultimately lead to the stimulation of cyclin/CDK complexes.

In addition to the injection experiments described above, other types of studies, such as cyclin D1 elevation by ras(13) or cyclin D1 promoter activation by ras(14) , make it clear that proto-oncogene signaling molecules must be involved in controlling cyclin-related proteins, although how this occurs is not well understood yet. This study has identified a reagent which is likely to provide important information in the search for this connection. The evidence presented here indicates that PGA, a member of the growth-inhibitory cyclopentenone prostaglandin family(15) , does not interfere with the action of any of the proto-oncogene signaling molecules listed above, whereas it does block the activation of G phase cyclin/CDK complexes and the hyperphosphorylation of pRb. The fact that its inhibition can be overcome by E2F-1/DP-1 injection or by adenoviral E1A protein injection clearly indicates that it does not block any enzymatic function required for DNA synthesis. These are the characteristics predicted for an inhibitor whose target is closely related to the connection point between the action of signaling molecules and cell cycle regulatory proteins.

EXPERIMENTAL PROCEDURES

Materials

Cell Culture

Flow Cytometry

Preparation and Microinjection of Oncoproteins

MAP Kinase and Cyclin-dependent Kinase Assay

Western Blotting

RESULTS

To determine an inhibitory range of PGA, rapidly growing cultures of NIH 3T3 cells were treated with various concentrations for 24 h, labeled with [H]thymidine for 4 h at the end of the PGA treatment, fixed, and autoradiographed. Parallel cultures were treated similarly except that, after 24 h, the PGA was removed and replaced with normal medium containing [H]thymidine for an additional 24 h, to determine reversibility. Concentrations of PGA higher than 20 µM efficiently blocked thymidine incorporation, while concentrations of 25 µM or less were found to be reversible (Fig. 1A). Reversible inhibition was observed even after 48 h of PGA treatment, although continued inhibition required addition of fresh PGA every 24 h (data not shown). Concentrations above 35 µM were cytopathic.

Figure 1: Reversible inhibition of NIH 3T3 and v-raf transformed cells by PGA. A, rapidly growing NIH 3T3 cells (circles) or v-raf transformed NIH 3T3 cells (triangles) were treated with various concentration of PGA for 24 h. For the last 4 h, the cells were labeled with [H]thymidine (open symbols). In parallel plates, after 24 h of PGA treatment, the cells were washed 3 times with fresh medium containing no PGA and cultured for another 24 h in the presence of [H]thymidine (closed symbols). After autoradiography, the labeling index was determined by counting more than 200 cells. The error bars indicate standard error (n = 2). B, rapidly growing NIH 3T3 cells were treated either with 1 mM hydroxyurea (squares) or 25 µM PGA (open circles), or ethanol, the solvent used for PGA treatment (closed circles). The cells were pulsed with [H]thymidine for 1 h at the indicated times after addition of chemicals. After autoradiography, the labeling index was determined by counting more than 200 cells. Similar results were obtained in two separate experiments.

Cell Cycle Considerations

To further analyze the point at which PGA inhibits cell cycle progression, asynchronously growing cultures were treated with PGA for 24 h, and their DNA content was analyzed by flow cytometry. In accordance with previously reported data, it was apparent that blockage at multiple points takes place(28) . Such cultures exhibited an increased proportion of the cells in both G and G/M phases, while the number of the cells in S phase was reduced (Fig. 2, A and B).

Figure 2: PGA arrested the cell cycle at G and G/M. The DNA content of the cell was analyzed by flow cytometry monitoring the fluorescence intensity of the propidium iodide-stained cells. A-G, NIH 3T3 cells; H and I, v-raf-transformed NIH 3T3 cells. A, rapidly growing cells; B, cycling cells treated with 25 µM PGA for 24 h; C, serum-starved quiescent cells (G arrested population); D, serum-starved cells were stimulated with 10% serum for 15 h (synchronous S-phase population); E, quiescent cells were treated with 25 µM PGA in the presence of 10% serum for 15 h; F, serum-starved cells were stimulated with 10% serum for 24 h; G, PGA was added to synchronous S-phase cells for 9 h; H, rapidly growing v-raf-transformed NIH 3T3 cells; G, v-raf-transformed NIH 3T3 cells treated with 25 µM PGA for 24 h.

In order to confirm that cell cycle blockage can take place in either G or G/M phase, synchronized cultures were treated with PGA. When the culture was synchronized in G by serum deprivation (Fig. 2C) prior to treatment with PGA and stimulation with serum, essentially all the cells were blocked in G as indicated by their DNA content (Fig. 2E). Control cultures stimulated with serum in the absence of PGA progressed into S phase (Fig. 2D). Cultures treated with PGA at 15 h after serum addition, when most cells were in S phase (Fig. 2D), appeared in G/M phase after 24 h (Fig. 2G). The fact that most of the S phase cells progressed into G/M phase in the presence of PGA confirms the previous results indicating that PGA does not block the progress of an ongoing S phase. The fact that few of these cells had progressed through mitosis into G, as would have been the case in the absence of PGA (Fig. 2F), indicated the presence of a G/M phase block by the inhibitor. There was, however, a clear distinction between G and G/M blockages. While the G arrest could be maintained for 48 h by replenishing the PGA-containing medium every 24 h, the majority of G/M arrested cells, in the presence of PGA, eventually went through the mitosis and were blocked in G at 20 h after the addition of PGA to synchronous S phase cells (data not shown). This indicates that PGA-induced G/M blockage is transient or that PGA treatment results in the elongation of time required for progression through G/M phases. The blockage at two different cell cycle points may indicate that PGA targets a molecule(s) important for the regulation of cell cycle progression at multiple points.

Finally, the point in G at which PGA exerts its inhibition was determined. For this experiment, NIH 3T3 cultures were synchronized in G by serum deprivation for 48 h. Serum and [H]thymidine were then added to the cultures. In one set of such cultures, PGA was added to separate dishes at various times after serum addition. These cultures were all fixed at 24 h after the initial addition of serum. If the cells failed to become labeled, it would indicate that the added PGA had blocked entry into S phase. As an indication that PGA functions late in G, it was found that even when it was administered 6 h after serum addition, PGA was efficiently able to block DNA synthesis. The efficiency of inhibition was reduced when PGA was added later than 6 h after serum addition, and, at 10 h, little inhibition was observed (Fig. 3). To determine the normal time of entry into S phase, a parallel set of quiescent cultures received serum and thymidine, but no PGA. These cultures were fixed at various times after the addition of serum. In these cultures, the cells became labeled with thymidine beginning 10 h after serum addition until reaching a maximum at 15 h. This indicates that S phase began between 10 and 15 h after the addition of serum to these cultures. It is not known how long it takes PGA to exert its inhibitory effects after being added to a culture, but these data indicate that it is inhibitory as long as it is added approximately 3 h prior to the onset of DNA synthesis. This result is consistent with its execution point at or near the restriction point late in G at which a cell becomes committed to enter S phase and complete another cell cycle.

Figure 3: PGA-induced G arrest is localized to late G phase. NIH 3T3 cells were made quiescent by serum starvation. To determine the time course of entry into S-phase after serum restimulation, the cells received 10% serum and [H]thymidine together and were fixed at various times there after (closed circles). To locate the PGA blockage point, cultures received serum and [H]thymidine simultaneously but were treated with 25 µM PGA at various times after the serum addition (open circles). These cultures were fixed at 24 h after the serum addition. In this case, labeled cells indicate the failure of the PGA treatment to block the cells in the G cell cycle phase. Labeling index was determined by counting more than 200 cells after autoradiography (n = 3).

The Effects of PGA on Proliferative Signaling Molecules

Figure 4: Microinjection studies. NIH 3T3 cells on coverslips were made quiescent by serum starvation for 48 h. Quiescent cells then received medium containing 10% calf serum and 25 µM PGA and were cultured for 12 h. All the cells inside the circle, drawn on the back of the coverslip (approximately 100-200 cells), were injected with the protein(s) indicated (solid bars in PGA section). Injected proteins include Leu-61 Ras , E2F-1, DP-1, E2F-1/DP-1, and E1A. As positive controls, serum-starved (SS) quiescent NIH 3T3 cells were injected (solid bars in SS section). The noninjected cells outside the circle served as controls for each injection (shaded bars). After microinjection, cells were labeled with [H]thymidine for 20-24 h, and the labeling index was determined by autoradiography. For the noninjected cells, more than 200 cells were counted, and for injected cells, the mean value of counted cells was 125 with the minimum counting of 80 cells. The number of experiments for each injection is indicated by numbers (n =) in the graph.

To confirm the observation that PGA interferes with proliferative signaling at a point subsequent to the action of cellular Ras, the action of two well characterized downstream targets of Ras activity were analyzed. The effect of PGA upon v-raf-transformed cells was first examined. A number of biological and biochemical observations confirm that biologically active cellular Ras proteins induce the activation of cellular Raf proteins. When PGA was added to NIH 3T3 cells transformed by oncogenic raf, thymidine incorporation was blocked in a dose-dependent and reversible manner (Fig. 1A). In addition, when raf-transformed cells were treated with PGA and subjected to FACS analysis 24 h later, the cells were seen to be inhibited in G and G/M (Fig. 2, H and I), as was observed with nontransformed NIH 3T3 cells. The fact that raf-transformed cells were effectively inhibited in cell cycle progression by PGA is consistent with the idea that PGA inhibits a molecule whose function is required subsequently to the action of cellular Raf protein.

The biological studies were extended with biochemical analysis of MAP kinase, a molecule whose activity is stimulated by both Ras and Raf activity, and which therefore functions downstream of both in proliferative signaling. The activity of MAP kinase was assessed by immunoprecipitation with specific anti-ERK2 antibodies followed by incubation with a substrate, myelin basic protein, in the presence of [-P]ATP. The amount of the myelin basic protein phosphorylation indicates the level of MAP kinase activity in the cell at the time of immunoprecipitation. Addition of serum to serum-deprived cells results in a rapid, more than 10- to 20-fold, increase in the activity of MAP kinase compared to quiescent cells (Fig. 5, lanes 2 and 3). When PGA was added together with serum or when the cells were pretreated with PGA for 2 h prior to the addition of medium containing serum and PGA, the activation of MAP kinase was not altered (Fig. 5, lanes 4 and 5). PGA itself has little effect on the basal MAP kinase activity (Fig. 5, lanes 1 and 2). The fact that neither injected Leu-61 Ras nor transformation with oncogenic raf is able to override PGA inhibition, together with the fact that serum-induced activation of MAP kinase is not altered by PGA treatment, clearly indicates that it blocks an activity which is essential to proliferation but which is not apparently involved in the action of several well characterized proto-oncogene components of the proliferative signal transduction pathway.

Figure 5: Serum-induced MAP kinase activation was not inhibited by PGA treatment. Serum-starved quiescent NIH 3T3 cells were harvested after treatment with 25 µM PGA for 2 h (lane 1), nothing (lane 2), 10% serum and solvent for 5 min (lane 3), 10% serum and 25 µM PGA for 5 min (lane 4), or 25 µM PGA pretreatment for 2 h followed by 10% serum and 25 µM PGA for 5 min (lane 5). Cell lysates were made and MAP kinase was immunoprecipitated using anti-ERK2 antibody. The kinase activity in the immunoprecipitate was determined using myelin basic protein as a substrate. Similar results were obtained in all three such experiments performed.

Microinjection of Adenoviral E1A or E2F-1/DP-1 Transcription Factors

()

Although E2F-1 microinjection induced S phase in serum-starved cells, it failed to efficiently override the PGA G arrest (Fig. 4). Since DP-1 is another transcription factor which associates with E2F-1 and potentiates the E2F transcription activity (7) , we tested the S phase inducing activity of DP-1 protein. While microinjection of DP-1 alone could induce S phase in serum-starved cells, it also failed to override PGA blockage. Combined microinjection of E2F-1 and DP-1, however, was able to override the PGA growth arrest efficiently. As a biological marker of proliferative control which is somewhat upstream of E2F/DP-1 activity, we next microinjected E1A protein. When purified E1A protein was microinjected into NIH 3T3 cells treated with PGA, the cells were efficiently induced to incorporate labeled thymidine (Fig. 4). Because E2F-1/DP-1 and the modulation of the activity of the molecules targeted by E1A were able to overcome PGA inhibition, this inhibition must involve molecules required for the control of proliferation rather than a metabolic process required for DNA synthesis. Furthermore, it is clear that PGA inhibits a step involved in cell cycle control which functions prior to the action of the targets of E1A and the release of E2F/DP-1 transcription factor.

Blockage of pRb Hyperphosphorylation and Cyclin/CDK Activation

Figure 6: The hyperphosphorylation of pRb was inhibited by PGA treatment. Serum-starved NIH 3T3 cells (lane 1) or serum-starved cells stimulated with serum and solvent (lane 2) or with serum and 25 µM PGA (lane 3) were analyzed. After 12 h of culture (at early S phase; see Fig. 4.), cells were lysed and pRb was immunoprecipitated. The electrophoretic mobility of pRb was monitored by Western blotting. Fast migrating hypophosphorylated pRb (Hypo-P-pRb) and slowly migrating hyperphosphorylated pRb (Hyper-P-pRb) were detected as well as IgG heavy chain (IgG HC) and light chain (IgG LC) which were used for immunoprecipitation. Similar data were obtained in two separate experiments.

There is extensive evidence that pRb phosphorylation requires the activation of cyclins and their associated kinases. Cyclin D/CDK4 becomes an active kinase in mid to late G and has the ability to phosphorylate pRb directly(32) . Later in G, apparently near the restriction point, cyclin E/CDK2 becomes active. The effect of PGA upon these two kinases was, therefore, analyzed. NIH 3T3 cells were deprived of serum for 48 h prior to the addition of serum alone, or serum and PGA together. These cells were cultured for an additional 10 h and lysates were prepared. From these lysates, immunoprecipitates were made with antibodies against CDK2, CDK4, cyclin D, or cyclin E. The immunoprecipitates were then incubated with the appropriate substrate to detect kinase activity; recombinant pRb in the case of CDK4 and cyclin D or histone H1 in the case of CDK2 and cyclin E. The phosphorylated substrates were then separated by SDS-PAGE and autoradiographed. The activity of both kinases was low in quiescent cells. The added serum stimulated the ability of the cyclin D/CDK4 complex to phosphorylate pRb as indicated either in the cyclin D or in the CDK4 immunoprecipitate. In the presence of PGA, however, the kinase activity in each immunoprecipitate was equal to or even less than that seen in quiescent cells (Fig. 7A). In addition, the activity of the cyclin E/CDK2 complex was inhibited by added PGA. As above, the added serum greatly stimulated the activity of this complex as assessed in immunoprecipitates with either cyclin E or CDK2, while no stimulation was seen in cells treated with serum and PGA together (Fig. 7B).

Figure 7: Activation of G CDKs were abolished by PGA. Control cells were serum-starved for 48 h without any treatment (lanes 1 and 4). Serum-starved NIH 3T3 cells were either stimulated with serum and solvent (lanes 2 and 5) or serum and 25 µM PGA (lanes 3 and 6). After 10 h of culture (late G phase, see Fig. 4), cells were lysed and CDK4 (A, lanes 1-3) or cyclin D1 (A, lanes 4-6), CDK2 (B, lanes 1-3), or cyclin E (B, lanes 4-6) were immunoprecipitated using respective antibodies. The kinase activity associating with the precipitate was determined using recombinant pRb (for CDK4 and cyclin D1) or histone H1 (for CDK2 and cyclin E). The result was reproducible in 2 (for CDK4, cyclin D1, and cyclin E immunoprecipitation) or in 4 (for CDK2 immunoprecipitation) separate experiments.

Expression of p21 Protein

Figure 8: Induction of p21 protein by PGA2. A, rapidly growing NIH 3T3 cells were treated with solvent ethanol as control (lane 1), 25 µM PGA (lane 2), O gray of mock irradiation (lane 3), or 5 gray of -irradiation (lane 4). Cells were harvested after 6 h of PGA treatment or 6 h after the irradiation. B, serum-starved NIH 3T3 cells received serum and solvent (lanes 5 and 7) or serum and 25 µM PGA (lanes 6 and 8). Cells were harvested 6 h (lanes 5 and 6) or 9 h (lanes 7 and 8) after the serum addition. Each cell lysate containing 50 µg of protein was analyzed by Western blot for p21 protein. The result was reproducible in 2 separate experiments.

DISCUSSION

Although its mechanism of action is unknown, pharmacological studies suggest that PGA is taken up by a carrier system at the plasma membrane. After internalization, PGA binds to cytosolic proteins and moves to the nucleus(35) . Cell cycle blockage in mid to late G has been well documented(36) . In this study, the blockage by PGA in NIH 3T3 cells was confirmed to be in late G, within 3 h of the initiation of DNA synthesis. This observation suggests that PGA might be interacting with a molecule (molecules) critical for passage through the restriction point just prior to S phase. In addition, a blockage in G/M is also observed as reported previously(28) . To confirm these results, cells were synchronized in either G or S phase at the time of PGA addition. In such cultures, cells became blocked either in G or in G/M, respectively, although blockage in G/M was transient. No evidence for blockage in S phase was obtained. It, therefore, appears likely that PGA targets a molecule(s) required for cell cycle progression both in late G and in G/M phase.

Analyses were performed to localize PGA inhibition in relationship to well characterized molecules involved in cell proliferation. The fact that microinjected oncogenic Ras was unable to overcome the inhibitory effects of PGA treatment indicates that PGA directly targets Ras, a molecule required downstream of Ras action, or a molecule involved in an entirely separate pathway required for cell cycle progression from G to S phase. This result is the opposite of that observed with TGF-. In mink lung epithelial cells, the proliferation inhibitory action of TGF- was completely overcome by injection of oncogenic Ras(30) . These results were extended by demonstrating that NIH 3T3 cells transformed by oncogenic raf were also inhibited by PGA treatment. This observation indicates PGA apparently acts downstream of Ras and even downstream of Raf. When E2F-1 and DP-1 or adenoviral E1A protein was injected into PGA-treated cells, however, efficient entry into S phase was observed. The results reported here indicate that the target(s) of PGA action functions between these two markers of proliferative induction, Ras/Raf and the activity of cellular molecules triggered by E1A or E2F-1/DP-1.

A number of treatments are known to block cell cycle progression in mid to late G. In Balb/c 3T3 cells, treatment with sodium butyrate or a combination of the ion channel blockers amyloride and bumetanide efficiently block cell cycle progression in mid-G at a point which is prior to the requirement of Ras in late G(31) . Interestingly, the effects of these inhibitors were not overcome by injection of either oncogenic Ras or E1A. We interpret this observation to indicate that these two inhibitors block metabolic processes essential during preparation for cell cycle transit, processes such as the duplication of important cellular components. PGA, on the other hand, apparently interferes with signaling or cell cycle control molecules which directly regulate transition between different phases of the cell cycle, leaving all preparatory metabolic requirements unaffected as indicated by the fact that E2F-1/DP-1 or E1A could overcome PGA inhibition.

A number of biochemical markers of proliferative signaling and cell cycle control were analyzed next. MAP kinase, whose activity is stimulated by the Ras-Raf-MEK pathway, was shown to be unaffected by PGA treatment, while the kinase activity of both cyclin D/CDK4 and cyclin E/CDK2 were completely inhibited, as was the hyperphosphorylation of pRb. Although it is p130, not pRb, which is primarily associated with E2F in G phase of mouse cells (37) , there are several reasons to believe that pRb functions as one of the regulators of E2F in these cells and can serve as an indicator of the activity of the class of E2F regulating proteins. It has recently been shown that dihydrofolate reductase, an enzyme whose expression is regulated by E2F, is expressed at higher levels in pRb negative murine cells than normal cells(38) . In addition, over expression of cyclin D in murine cells, which presumably induces hyperphosphorylation of pRb (6) , can induce the activity of a reporter gene controlled by an E2F promoter(39) . The facts that PGA blocks the hyperphosphorylation of pRb, and that E2F-1/DP-1 or E1A microinjection overcomes PGA-induced growth arrest, are taken to indicate that PGA ultimately blocks the normal release of E2F/DP-1.

A possible explanation for the inhibition of G phase cyclin-CDK kinase activity by PGA2 is the induction of p21 protein, since this protein has been shown to inhibit both CDK4 and CDK2 activity(33) , and its binding to CDK2 prevents the activating phosphorylation by CDK activating kinase(40) . In this study we have shown that PGA-induced G arrest was not overridden by oncogenic Ras. On the other hand, although TGF- also induces CDK inhibitory proteins expression levels including p21(41) , Ras can override TGF--induced G arrest(30) . A possible interpretation of these observations is that interruption of mitogenic signaling at any site along the pathway, upstream of Ras in the case of TGF- and downstream of Ras in the case of PGA, may result in the up-regulation of CDK inhibitory protein(s). If so, p21 induction might be a consequence rather than a cause of inhibition of the proliferative process. On the other hand, the possibility that PGA directly induces p21 synthesis is supported by the fact that other genes are reported to be induced by PGA treatment, including gadd153, heat shock proteins, and hemoxylase (42, 43, 44) .

We conclude that PGA is targeting downstream of most elements of the Ras-Raf pathway and elements prior to or at the site of the activation of most cyclins and associated kinases. Consequently, PGA apparently interrupts an activity which might be closely related to the point at which these two critical cell proliferation control mechanisms (proliferative signaling and cell cycle regulation systems) interact. Interestingly, however, microinjection of either E2F-1 or DP-1 alone could not efficiently override PGA cell cycle arrest while each was able to induce S phase efficiently in serum-starved, quiescent cells. This may indicate the possibility that PGA blocks the cell cycle not only by disrupting the connection between the two proliferation control systems described above, but also by affecting the interaction of the cell cycle controlling system and the DNA synthesis process, where pRb, pRb-related proteins, and E2F/DP transcription factors are involved(9) . Another possible explanation is that by suppressing the cyclin E-CDK2 activity, PGA can shut off the positive feed back loop of cyclin E, pRb, and E2F proposed by DeGregori et al.(45) , which may result in the insufficient release of free E2F/DP transcription factor. On the other hand, since the E2F-1/DP-1 complex has more potent transactivation activity than each protein alone(7, 46) , E2F-1/DP1 combined microinjection may readily be able to induce the molecule required for S-phase without this cyclin E-pRb-E2F amplifying loop.

In this study we have not tested the possible involvement of PGA in several other pathways with potential to modulate the activity of the cell proliferation controlling molecules discussed above. For instance, cyclic AMP is inhibitory in many cell types, and it has been suggested that PGA might function by elevating the intracellular concentration of cyclic AMP. Since antiproliferative prostaglandins have been reported to arrest cell growth without cyclic AMP elevation(47) , cyclic AMP may not be a mediator of PGA growth inhibition. Furthermore, recent studies suggest that cyclic AMP exerts its cell growth inhibitory effect by protein kinase A-dependent phosphorylation of either the regulatory domain or the kinase domain of Raf protein, resulting in either disruption of the association between Ras and Raf (48, 49, 50) or inhibition of the Raf kinase activity including v-Raf(51) , respectively. Since MAP kinase activation requires Raf activity(48, 50, 52) , and since PGA2 did not inhibit the serum-induced MAP kinase activation, neither of cyclic AMP-dependent Raf inactivation mechanisms appears to be involved in PGA growth arrest.

While it is unlikely that PGA functions through cyclic AMP, there are numerous signaling systems functioning within the cell which affect proliferation. Some of these, such as the stress kinase family, are likely to affect proliferative signaling(53) . Others, such as small GTP-binding proteins of the Rho family, are likely to be required for proliferation in some cells(54) . Finally, the cytokine signaling pathway might function either to enhance or interfere with proliferative signaling. The evidence above makes it clear that PGA affects a molecule closely related to the point of connection between the oncogene signaling pathway and the action of cyclins and related proteins. This might be accomplished by its ability to interact directly with a molecule whose action is closely related to the linkage between these two cell proliferation control systems, or it might be due to its ability to modify a separate signaling system which ultimately affects such a linkage molecule(s). Our data raise the possibility that p21 might be one of such linkage molecules. In any case, it is likely that PGA will provide a valuable tool in unraveling the important mechanism by which the signal from proto-oncogenes alters the activity of cyclins and their associating proteins, thereby directly controlling passage through the cell cycle.

FOOTNOTES

*

This investigation was supported by National Institutes of Health Grants GM52271 and CA53496 (to D. W. S.) and by American Cancer Society Grant CB-39 (to S. W. H.).The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Dept. of Molecular Biology, NC2-150, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195-5245. Tel.: 216-444-0892; Fax: 216-444-0512; hitomim{at}cesmtp.ccf.org.

()

The abbreviations used are: MAP kinase, mitogen-activated protein kinase; CDK, cyclin-dependent kinase; pRb, product of retinoblastoma susceptible gene; PGA, prostaglandin A; ERK, extracellular signal-regulated kinase; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; TGF-, transforming growth factor .

()

D. W. Stacey and S. W. Hiebert, unpublished results.

ACKNOWLEDGEMENTS

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